1. Field of the Invention
This invention relates to a method and apparatus for testing Mycobacteria avium-intracellulare ("MAI") for antimicrobial agent sensitivity.
2. Description of the Prior Art
Human immunodeficiency virus type 1 or HIV causes acquired immunodeficiency syndrome ("AIDS") which is a fatal disease approaching epidemic proportions throughout the world. By current estimates, about 15-34% of infected individuals will probably develop AIDS within 3-5 years. During the asymptomatic stage of AIDS, although most patients have no symptoms, some patients a few weeks after exposure develop a disorder resembling mononucleosis. Later, its symptoms include fatigue, fever and swollen glands, diarrhea and minor infections. Most of these symptoms disappear initially, but may recur. When AIDS develops, it is usually characterized by a major opportunistic infection, such as Pneumocystis pneumonia, or an opportunistic tumor, such as Kaposi's sarcoma or a lymphoma. At this stage, the disease is uniformly fatal.
It has been found that more than 50% of the AIDS patients have MAI present in their bodies. Wallace, J. M. and Hannah, J. B., "Mycobacterium avium Complex in Patients with Acquired Immunodeficiency Syndrome--A Clinicopathologic Study", Chest 93(5): 926-932 (1988). The MAI complex infection in AIDS patients has been shown to be widely disseminated in the patient, however the most common source of isolation is in the blood. There is currently no effective treatment against MAI as these organisms are often resistant to standard therapy.
It is known to use isolation techniques for determining the presence or absence of MAI in the patient's blood. One method involves using the BACTEC Radiometric System, which is a product of the Johnston Division of Becton and Dickenson. The system itself utilizes hemoculture tubes that contain Middlebrook 7H12 liquid broth plus 0.05% (v/v) sodium polyanethyl sulphonate in hemoculture vials. In addition, the 7H12 broth contains Carbon-14 labeled palmitic acid. In use, vials containing mycobacterial growth give off Carbon-14 labeled CO.sub.2 and this is detected by a device similar to that used for liquid scintillation counter capable of detecting beta emitters. See Kirihara, J. M. et al. "Improved Detection Times for Mycobacterium avium Complex and Mycobacterium tuberculosis with BACTEC Radiometric System", J. Clin. Microbiol. 22:841-845 (1985).
Another method of isolation involves using genetic probes which rely upon DNA hybridization. Gonzalez, R. et al., "Evaluation of Gen-Probe DNA Hybridization Systems for the Identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare Diagnosis", Microbiology Infect. Dis. 8:69-77 (1987).
These known methods, although effective, require expensive equipment and specialized operating personnel and materials. Thus, smaller hospital centers where few AIDS patients are seen, field laboratories, and third world countries, where resources are limited, do not have this specialized equipment and personnel. A simpler and more inexpensive method and apparatus of isolating and identifying MAI would be of substantial benefit in such situations.
It is known that many atypical Mycobacteria grow on basal salt media devoid of any carbon sources other than paraffin wax which is introduced into the media in the form of paraffin wax coated rods. Fuhs, G. W., "Der Mikrobielle Abbau Von Kohlenwasserstoffen", Arch. Mikrobiol. 39:374-422 (1961). Mishra, S. K. et al., "Observations On Paraffin Baiting As a Laboratory Diagnostic Procedure in Nocardiosis", Mycopathologica and Mycologia Applicata 51 (2-3):147-157 (1973) utilized paraffin coated rods and basal salt medium to isolate Nocardia asteroides from clinical specimens such as sputum, bronchial lavage and cerebrospinal fluid.
The technique was further improved by substituting paraffin wax coated slides for rods and thereby making possible the use on an in situ Kinyoun cold acid-fastness staining procedure for organisms growing on the paraffin coated slide. Ollar, R. A., "A Paraffin Baiting Technique that Enables a Direct Microscopic View of "in situ" Morphology of Nocardia asteroides with the Acid-Fast or Fluorescence Staining Procedures", Zbl. Bakt. Hyg., Abt. Orig. A, 234:81-90 (1976). With this assay, a positive reaction tells the user immediately that a mycobacteria organism other than M. tuberculosis is present.
Once MAI is identified, there remains a need to test the MAI for antibiotic sensitivity. U.S. Pat. No. 3,826,717 provides an antibiotic sensitivity test container which includes a plurality of wells which contain a solid nutrient media. The wells are positioned in rows with each row containing a single antibiotic and different wells within the row having different concentrations. A control well is provided which contains no antibiotic but the media.
Despite the above teachings, however, there still remains a need for efficient and economical method and an inexpensive apparatus for testing MAI for antimicrobial agent sensitivity.